Protein Cage
The protein cage is the most crucial component in our project's detection module, which is based on the 2-keto-3-deoxy-phosphogluconate (KDPG) aldolase from the Entner−Doudoroff pathway of the hyperthermophilic bacterium Thermotoga maritima.Considering that as a detection module on the test paper, it needs high stability, we want to do some research through dry experiment.
Mi3 subunit
We first performed 50ns molecular dynamics simulations at 298.15K,323.15K and 348.15K for the subunit of Mi3. Using the Charmm36 force field, TIP3P was selected for the water model.
Fig. 1. Simulation data for Mi3 subunit at 298.15K,323.15K,348.15K for 50 ns.
The RMSD analysis revealed minimal structural alterations in the Mi3 subunit during simulations at 298.15K and 323.15K over a duration of 50ns. However, at 348.15K, there was a slight fluctuation observed in the Mi3 subunit. Furthermore, RMSF analysis indicated that residues 131 to 141 exhibited higher flexibility, providing insights into the protein's dynamic behavior.In terms of radius of gyration (Rg), the data at 298.15K and 323.15K exhibited comparable values, suggesting similar compactness in the protein structure under these conditions. Conversely, at 348.15K, the Mi3 subunit displayed significant fluctuations initially, ultimately stabilizing at a lower level.The solvent-accessible surface areas of all three temperatures were found to be quite similar.
Fig. 2. Secondary structure analysis data for Mi3 subunit at 298.15K,323.15K,348.15K for 50 ns.(A)The ratio of the number of amino acid residues to total amino acids that form conventional secondary structures at three temperatures.(B)The ratio of the number of amino acid residues forming the α-Helix to the total amino acidsat three temperatures.(C)The ratio of the number of amino acid residues to the total amino acids that form the β-Sheet at three temperatures.
Analysis of the secondary structure showed that the data of Mi3 subunit at 298.15K and 323.15K were similar during the 50ns, while the proportion of amino acids forming secondary structure of Mi3 subunit at 348.15K decreased, mainly reflected in α-Helix. The self-assembly of the protein cage is dependent on the interaction of α-Helix between different subunits, indicating that the stability of the protein cage may be affected at 248.15K.
Mi3 subunit trimer
We further expanded the subject of our study by selecting the Mi3 subunit trimer located on one of the vertices of the protein cage for further analysis.
Fig. 3. Simulation data for Mi3 subunit trimer at 298.15K,323.15K,348.15K for 50 ns.
The RMSD at 298.15K and 323.15K was consistently around 0.2nm, and at 348.15K, it slightly increased but still remained relatively stable at about 0.3nm. This suggests that the protein trimer maintained a stable structure over the 50ns simulation at all three temperatures. The RMSF at the three temperatures showed a similar trend, with slightly higher values at 348.15K. The Rg values also indicated a high compactness of the trimer throughout the 50ns simulation at all temperatures. Analysis of solvent accessible area further confirmed a decrease in protein stability with increasing temperature.
Fig. 4. Structure of Mi3 subunit trimer.
The solvent accessible area of the trimer exhibited a reduction compared to that of individual subunits, implying a tight binding among the proteins. Moreover, a comparative analysis revealed a notable decrease in flexibility for residues 131-141 within the trimer, suggesting that trimer formation played a stabilizing role in this specific region of the structure.
Fig. 5. Secondary structure analysis data for Mi3 subunit trimer at 298.15K,323.15K,348.15K for 50 ns.
At 348.15K, the Mi3 subunit trimer exhibited a reduction in the number of amino acid residues forming secondary structures. The effective structure diminished by approximately 5% over the simulation time, with a significant reduction observed in the major α-helix structure.
Mi3
We finally simulated the overall structure of Mi3, and due to the limitation of computational power, we only considered the case in this limit of 373.15K.
Their partial trajectories are animated as follows：
Fig. 6. Trajectory Animation of Mi3 at 298.15K
Fig. 7. Trajectory Animation of Mi3 at 373.15K
Fig. 8.Simulation data for Mi3 at 298.15K,373.15K for 25 ns.
The RMSD analysis indicates that the structure gradually stabilizes and reaches a stationary state as the simulation progresses. Examination of the solvent accessible area suggests that the protein cage adopts a more compact structure at 298.15K. This observation is further supported by the calculated Radial Distribution Function (RDF), where the image broadens and the Radius of Gyration (Rg) increases at 373.15K, indicating a less compact structure.
Fig. 9. Secondary structure analysis data for Mi3 at 298.15K,373.15K for 25 ns.
At 373.15K, the overall structure percentage of Mi3 is reduced by approximately 4%. Specifically, there is a significant reduction in the percentage of α-Helix, while the percentage of β-sheet remains relatively consistent. Additionally, there is a noticeable increase in the percentage of random coil structures. These changes collectively suggest a gradual reduction in the ordered structure of Mi3 at 373.15K.
We employed molecular dynamics simulations to assess the stability of Mi3 in its monomeric, trimeric, and complete structural forms. The multimerized state was observed to enhance stability, particularly in large flexible regions of the protein cage. Notably, the parameters of Mi3 at 298.15K and 323.15K exhibited close similarity, indicating that the protein cage maintained stability within this temperature range. This finding suggests its suitability for utilization as a component of the detection module.
We conducted molecular dynamics simulations on the entire Mi3 structure. However, due to computational limitations, simulations were carried out at only two temperatures and restricted to a timescale of 25 nanoseconds. In the future, we aim to leverage more potent computational servers to conduct more detailed calculations for a comprehensive evaluation of the protein cage's stability. This will empower us to optimize the original protein cage structure and swiftly assess alterations in its properties upon interaction with other molecules through computational modeling.
